Project description:To define chromatin structure changes along the yeast genome using microarrays. Nucleosomal DNA from WT and delta isw2 yeast were hybridized and differences in signals were calculated. Keywords: Nucleosomal DNA hybridization
Project description:Nucleosome positions were determined in wild type cells, cells lacking Isw2 or Ume6, and cells containing a hybrid Chd1-Ume6 chimeric remodeler
Project description:Nucleosome positions were determined in wild type cells, cells lacking Isw2 or Ume6, and cells containing a hybrid Chd1-Ume6 chimeric remodeler Matched MNase digests from W303 strain variants during log growth (OD600=0.4-0.6) were subject to paired-end sequencing for nucleosome mapping. For effects of the engineered fusion remodeling protein, a catalytically inactive (ATPase dead D513N) variant was included as a control.
Project description:It has previously been shown that Isw2 represses cryptic antisense transcripts from the 3’-end of three genes. However, whether Isw2 generally functions to repress cryptic RNA transcription is currently unknown. We thus sought to determine the loci at which Isw2 is required to repress cryptic non-coding RNA (ncRNA) expression and to map their transcription start sites relative to nucleosome positions on a global scale.
Project description:Histone octamers are thought to be a rigid part of nucleosomes that shape chromatin and block cellular machinery from accessing DNA. ATP-dependent chromatin remodelers like ISW2 mobilize nucleosomes to provide DNA access. We find evidence for histone octamer distortion preceding DNA being moved into nucleosomes and processive movement of the ATPase motor of ISW2 on nucleosomal DNA. DNA entering nucleosome is uncoupled from the ATPase activity of ISW2 and alterations of the histone octamer structure mediated by ISW2 by deletion of the SANT domain from the C-terminus of the Isw2 catalytic subunit. We also find that restricting histone movement by chemical crosslinking traps remodeling intermediates resembling those seen by loss of the SANT domain, further supporting the importance of changes in histone octamer structure early in ISW2 remodeling. Transient octamer distortions are stabilized by H3-H4 tetramer disulfide crosslinking, thereby linking intrinsic histone octamer flexibility to chromatin remodeling.
Project description:The distribution of nucleosomes along the genome is a significant aspect of chromatin structure and is thought to influence gene regulation through modulation of DNA accessibility. However, properties of nucleosome organization remain poorly understood, particularly in mammalian genomes where nucleosome positions have not been examined beyond isolated loci and promoter regions. Towards this goal we used tiled microarrays to identify stable nucleosome positions along the HOX gene clusters in human cell lines. We show that nucleosome positions exhibit sequence properties and long-range organization that are different from those characterized in other organisms. Despite overall variability of inter-nucleosome distances, specific loci contain regular nucleosomal arrays with 195bp periodicity. Moreover, such arrays tend to occur preferentially toward the 3’ ends of genes. Through comparison of different cell lines, we find that increased gene expression correlates with increased positioning of nucleosomes, suggesting an unexpected role for transcription in the establishment of well-positioned nucleosomes. Keywords: human chromatin nucleosome positions, nucleosomes, ChIP-chip
Project description:Chromatin remodelers regulate genes by organizing nucleosomes around promoters, but their individual contributions are obfuscated by the complex in vivo milieu of factor redundancy and indirect effects. Genome-wide reconstitution of promoter nucleosome organization with purified proteins resolves this problem and is therefore a critical goal. Here we reconstitute four stages of nucleosome architecture using purified components: Yeast genomic DNA, histones, sequence-specific Abf1/Reb1, and remodelers RSC, ISW2, INO80, and ISW1a. We identify direct, specific and sufficient contributions that in vivo observations validate. First, RSC clears promoters by translating poly(dA:dT) into directional nucleosome removal. Second, partial redundancy is recapitulated where INO80 alone, or ISW2 at Abf1/Reb1sites, positions +1 nucleosomes. Third, INO80 and ISW2 each align downstream nucleosomal arrays. Fourth, ISW1a tightens the spacing to canonical repeat lengths. Such a minimal set of rules and proteins establishes core mechanisms by which promoter chromatin architecture arises through a blend of redundancy and specialization.